U.S. patent number 10,919,967 [Application Number 16/312,629] was granted by the patent office on 2021-02-16 for antibodies against mac-1.
This patent grant is currently assigned to ALBERT-LUDWIGS-UNIVERSITATFREIBURG, BAKER IDI HEART & DIABETES INSTITUTE HOLDINGS LTD.. The grantee listed for this patent is Albert-Ludwigs-Universitat Freiburg, Baker IDI Heart & Diabetes Institute Holdings Ltd.. Invention is credited to Karlheinz Peter, Dennis Wolf, Andreas Zirlik.
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United States Patent |
10,919,967 |
Zirlik , et al. |
February 16, 2021 |
Antibodies against Mac-1
Abstract
The present invention provides an isolated monoclonal antibody
or an antigen-binding portion thereof which a) binds to Mac-1, b)
specifically inhibits the interaction of CD40L with activated Mac-1
and c) does not induce integrin outside-in signaling.
Inventors: |
Zirlik; Andreas (Freiburg,
DE), Wolf; Dennis (Schopfheim, DE), Peter;
Karlheinz (Hawthorn East, AU) |
Applicant: |
Name |
City |
State |
Country |
Type |
Albert-Ludwigs-Universitat Freiburg
Baker IDI Heart & Diabetes Institute Holdings Ltd. |
Freiburg
Melbourne |
N/A
N/A |
DE
AU |
|
|
Assignee: |
ALBERT-LUDWIGS-UNIVERSITATFREIBURG (Freiburg, DE)
BAKER IDI HEART & DIABETES INSTITUTE HOLDINGS LTD.
(Melbourne, AU)
|
Family
ID: |
1000005364317 |
Appl.
No.: |
16/312,629 |
Filed: |
June 13, 2017 |
PCT
Filed: |
June 13, 2017 |
PCT No.: |
PCT/EP2017/064339 |
371(c)(1),(2),(4) Date: |
December 21, 2018 |
PCT
Pub. No.: |
WO2017/220369 |
PCT
Pub. Date: |
December 28, 2017 |
Prior Publication Data
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|
|
|
Document
Identifier |
Publication Date |
|
US 20190161550 A1 |
May 30, 2019 |
|
Foreign Application Priority Data
|
|
|
|
|
Jun 21, 2016 [EP] |
|
|
16175382 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
16/2845 (20130101); C07K 2317/55 (20130101); C07K
2317/34 (20130101); C07K 2317/76 (20130101); C07K
2317/32 (20130101); C07K 2317/70 (20130101); A61K
2039/505 (20130101); C07K 2317/33 (20130101) |
Current International
Class: |
C07K
16/00 (20060101); C07K 16/46 (20060101); C07K
16/28 (20060101); A61K 39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2 444 101 |
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Apr 2012 |
|
EP |
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2629787 |
|
Oct 2018 |
|
EP |
|
Other References
Zirlik et al. CD40 Ligand Mediates Inflammation Independently of
CD40 by Interaction With Mac-1. Circulation. 2007;115:1571-1580.
(Year: 2007). cited by examiner .
Okwor et al. Interaction of Macrophage Antigen 1 and CD40 Ligand
Leads to IL-12 Production and Resistance in CD40-Deficient Mice
Infected with Leishmania major. The Journal of Immunology, 2015,
195: 3218-3226. (Year: 2015). cited by examiner .
Altieri et al. A unique recognition site mediates the interaction
of fibrinogen with leukocyte mac-1 (CD11b/CD18) . JBC 265(21)
12119-12122, 1990. (Year: 1990). cited by examiner .
PCT/EP2017/064339--International Search Report, dated Aug. 30,
2017. cited by applicant .
PCT/EP2017/064339--International Written Opinion, dated Aug. 30,
2017. cited by applicant .
Hermann Blankenbach, "Generierung und Charakterisierung eines neuen
Liganden-und Akivitats-spezifischen Antikorpers zur Selektiven
Hemmung der CD40L/Mac-1 Interaktion", Jun. 27, 2016. cited by
applicant .
D. Wolf, et al., "Binding of CD40L to Mac-1's I-Domain Involves the
EQLKKSKTL Motif and Mediates Leukocyte Recruitment and
Atherosclerosis-But Does Not Affect Immunity and Thrombosis in
Mice" Circulation Research, vol. 109, No. 11, Nov. 11, 2011, pp.
1269-1279. cited by applicant.
|
Primary Examiner: Haddad; Maher M
Attorney, Agent or Firm: Curatolo Sidoti Co., LPA Sidoti;
Salvatore A. Trillis, III; Floyd
Claims
The invention claimed is:
1. An isolated monoclonal antibody or an antigen-binding portion
thereof which a) binds to Mac-1, b) specifically inhibits the
interaction of CD40L with activated Mac-1, c) does not bind to
non-activated Mac-1, and d) does not induce integrin outside-in
signaling, characterized in that it binds specifically to a peptide
having the sequence SEQ ID NO: 9 and that it comprises six CDRs
selected from the group consisting of SEQ ID NOs:2-4 and SEQ ID
NOs:6-8.
2. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the monoclonal antibody or an
antigen-binding portion thereof limits the expression of
inflammatory cytokines.
3. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the monoclonal antibody or an
antigen-binding portion thereof blocks leukocyte recruitment in
vitro and in vivo in intravital microscopy.
4. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the monoclonal antibody or an
antigen-binding portion thereof does not affect thrombotic and
hemostatic functions of Mac-1.
5. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the light chain has an identity of at
least 80% to the amino acid sequence of SEQ ID NO:1 and that the
heavy chain has at least 80% identity to the amino acid sequence of
SEQ ID NO:5.
6. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the light chain has the amino acid
sequence of SEQ ID NO:1.
7. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the amino acid sequence of the heavy
chain corresponds to SEQ ID NO:5.
8. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the monoclonal antibody or an
antigen-binding portion thereof is selected from the group
comprising F.sub.ab fragments, single chain antibodies, diabodies
and/or nanobodies.
9. A pharmaceutical composition comprising a pharmaceutically
active amount of an antibody or antigen-binding portion thereof of
claim 1.
10. The pharmaceutical composition according to claim 9 for the
treatment of inflammation.
11. The isolated monoclonal antibody or antigen-binding portion
thereof of claim 1, wherein the monoclonal antibodies or
antigen-binding portions thereof are humanized.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national stage application of International
Application No. PCT/EP2017/064339, filed 13 Jun. 2017, which claims
priority from European Patent Application No. 16175382.7, filed 21
Jun. 2016, which applications are incorporated herein by
reference.
In the past decades inflammation was identified as driving force of
many pathologies, including atherosclerosis, Type 2 Diabetes,
sepsis, myocardial infarction, autoimmune diseases and
neurodegenerative disease. Targeting the inflammatory response has
been proposed as major goal in these pathologies. However, a major
limitation of such strategies remains that the inflammatory
response is critical for regeneration, survival, and host defense.
A safe and reliable anti-inflammatory therapy therefore represents
a major medical need. This is illustrated by glucocorticoids,
potent inhibitors of inflammation that compromise the immune
response, or COX-2 inhibitors, which can suppress inflammation, but
exhibit detrimental effects on the cardiovascular system.
Inflammation is a process that involves recruitment of leukocytes
to the site of injury mediated by leukocyte integrins, such as
Mac-1 (.alpha..sub.M.beta..sub.2, CD11b/CD18). Mac-1 is a potent
adhesion factor, susceptible to rapid inflammatory activation by
conformational change that exhibits increased affinity to its
ligands resulting in rolling, firm adhesion, and transmigration of
leukocytes into inflamed tissue. Mac-1 is a powerful target in
cardiovascular disease and therapeutic or genetic inhibition of the
integrin and has been shown to be highly effective in preventing
atherosclerosis, neo-intima formation, and thrombotic
glomerulonephritis. Besides its role in inflammation, Mac-1 was
initially named CR (complement receptor) 3 by its ability to bind
complement factors, such as C3bi, reflecting its role in host
defense, wound healing, thrombosis, and various other myeloid cell
effector functions. This broad repertoire of effector functions is
realized by a broad expression on the myeloid lineage, including on
monocytes, macrophages, and neutrophils, but also on NK cells, and
to a smaller extent on activated lymphocytes. Its functional
diversity is furthermore reflected by promiscuous ligand binding to
a large repertoire of proteins and proteoglycans, including ICAM-1,
fibrinogen, fibronectin, heparin, GPIb.alpha., RAGE, endothelial
protein C-receptor (EPCR), and CD40L. It has been proposed that
integrin antagonism is a promising target in inflammation. However,
its role in host defense and thrombosis may limit its clinical
use.
CD40 ligand (CD40L) is a transmembrane molecule of crucial interest
in cell signaling in innate and adaptive immunity. It is expressed
by a variety of cells, but mainly by activated T-Iymphocytes and
platelets. CD40L may be cleaved into a soluble form (sCD40L) that
has a cytokine-like activity. Both forms bind to several receptors,
including CD40. This interaction is necessary for the antigen
specific immune response. CD40L binds also to different receptors
whereby Mac-1 (.alpha.M.beta.2) is one receptor whereby said
interaction plays a role in arterial neo-intima formation,
leukocyte recruitment and atherosclerosis, pathogenesis of
atherothrombosis, monocyte adhesion and neutrophil infiltration and
release of pro-inflammatory cytokines (IL-8, IL-6).
Mac-1 is a classical adhesion factor involved in a variety of
inflammatory pathologies. Despite its promoting effect on leukocyte
recruitment in atherosclerosis and peritoneal inflammation, Mac-1
targeted therapy is limited by various side effects, such as
impaired wound healing and host defense. This is further reflected
by the human Leukocyte Adhesion Deficiency (LAD), which is
characterized by a defect of the integrin Mac-1, LFA-1, and CD11c
in the .beta.-subunit that impairs host defense. Unspecific
attempts to therapeutically inhibit Mac-1 seem therefore not
favorable. To circumvent these limitations novel monoclonal
antibodies are provided that specifically target the binding of
CD40L to Mac-1's major ligand binding I-domain within the
am-subunit of the integrin. CD40L represents a biased agonist for
Mac-1, mediating its pro-inflammatory function by serving as
endothelial adhesion factor for CD40L, but not by activation of
outside-in signaling pathways. CD40L/Mac-1 binding does not
interfere with CD40L-CD40 or Mac-1-GP1balpha and Mac-1-ICAM-1
binding, suggesting unique binding epitopes on each of the protein
surfaces.
Integrins are major adhesion receptors that transmit signals
bidirectionally across the plasma membrane, playing significant
roles in diverse biological processes including immune response.
Integrins contain two non-covalently associated type 1
transmembrane glycoprotein .alpha. and .beta. subunits; each
subunit contains large extracellular domains, a single-spanning
transmembrane domain and short cytoplasmic domain. The ability of
the integrin's extracellular domain to bind ligands depends on an
open-extended confirmation of the .alpha.M subunit ("activation")
and regulates cell adhesion and signal transduction, both
outside-in and inside-out signaling. The present invention relates
to a specific modification of the interaction between CD40L and the
integrin .alpha..sub.M.beta.2 (Mac-1).
It has been found that inactivation of distinct integrin functions
involved in inflammatory, but not in regenerative or immune
pathways could be achieved by selectively blocking Mac-1's
interaction to specific ligands, while not affecting others.
Monoclonal antibodies, specifically targeting the EQLKKSKTL (SEQ ID
NO:9) binding motif in Mac-1, which we have demonstrated to be
required for binding to its adhesive, pro-inflammatory ligand CD40L
have been constructed.
The present invention provides therefore isolated monoclonal
antibodies or antigen-binding portions thereof, which inhibit the
recruitment of leukocytes without undesired side effects. Such
antibodies or antigen-binding portions thereof
a) bind to Mac-1,
b) specifically inhibit the interaction of CD40L with activated
Mac-1, and
c) do not induce integrin outside-in signaling.
In the course of the present invention monoclonal antibodies have
been constructed whereby the most preferred embodiment is the
antibody in the following designated as anti-M7. The sequence of
the antibody has been determined and the CDRs were identified. With
this information and computational and conventional binding studies
it is possible to provide suitable other antibodies or
antigen-binding fragments thereof, which are derived from this
antibody. Since antibody technology has gained much interest in the
therapeutic area there are several engineered antibody fragments
available which can be used in practice. The term "monoclonal
antibody or antigen-binding fragment thereof" is understood in a
broad sense and includes therefore not only the F.sub.ab fragments
but also single-chained Fv fragments (scFv), diabodies which may be
bispecific, bispecific single chain fragments, triabodies,
tetrabodies or minibodies. The sequence information provided herein
can also be used to produce nanobodies which are derived from
camelite immunoglobulins. Many of those structures are summarized
in the review article of Holliger et al. (Nature Biotechnology,
vol. 23, no. 9 (2005), pp 1126-1136).
It is a preferred property of the isolated monoclonal antibodies or
antigen-binding portions thereof that they bind to Mac-1, whereby,
however, the binding to the activated Mac-1 is preferred whereas
the antibody structures of the present invention should not bind to
non-activated Mac-1. A distinction between activated and
non-activated Mac-1 can be performed by quantifying the binding
kinetics as for example described by Li et al. (Journal of
Immunology (2013), pp 4371-4381).
Another preferred embodiment of the isolated monoclonal antibodies
or antigen-binding portions thereof of the present invention is
that they limit the expression of inflammatory cytokines.
Another preferred property of the isolated monoclonal antibodies or
antigen-binding portions thereof is that they block leukocyte
recruitment in vitro and preferably in vivo. Such blockage can be
observed and measured in intravital microscopy as shown in the
examples of the present application.
A further preferred embodiment of the monoclonal antibodies or
antigen-binding portions thereof is that the thrombotic and
hemostatic functions of Mac-1 are not effected. This can be
measured by using suitable in vivo experiments.
The preferred embodiment disclosed herein designated as anti-M7 was
produced as a monoclonal antibody in the mouse system. It is
well-known to the skilled person that monoclonal murine antibodies
cannot be used in the therapy of humans since after repeated
administration of such murine antibodies anti-mouse antibodies are
generated in the patient. Therefore, the monoclonal antibodies or
antigen-binding portions thereof are preferably humanized.
Humanization means that the mouse framework of the antibody is
replaced by a human framework structure of an antibody which has
high similarity to the mouse antibody. By using suitable
computational models further adaptations of the amino acid
structure can be made in order to reduce the mouse character of the
antibody. It has, however, to be checked whether the proposed
changes in the amino acid sequence reduce the binding strength of
the humanized antibody or antigen-binding construct. Only such
amino acid substitutions are performed which do not negatively
affect the binding properties and in particular the
specificity.
It is assumed that such modified antibodies or antigen-binding
portions thereof should comprise at least three CDRs. The CDRs are
disclosed and have SEQ ID NOs:2-4 and 6-8, respectively. In a more
preferred embodiment the monoclonal antibodies or antigen-binding
portions thereof according to the present invention comprise at
least four, more preferred five and in particular preferred six
CDRs having the sequences of SEQ ID NOs:2-4 and 6-8,
respectively.
The light chain of the anti-M7 antibody has the amino acid sequence
as provided in SEQ ID NO:1 and the heavy chain corresponds to SEQ
ID NO:5. As already explained above, in the course of humanization
amino acid sequence changes are introduced into the amino acid
sequence. In preferred embodiments the isolated monoclonal
antibodies or antigen-binding portions of the present invention
have a light chain which has an amino acid identity of at least
80%, preferred of at least 85%, more preferred of at least 90% and
in particular preferred of at least 95% identity to SEQ ID
NO:1.
In preferred embodiments the isolated monoclonal antibodies or
antigen-binding portions of the present invention have a heavy
chain which has an amino acid identity of at least 80%, preferred
of at least 85%, more preferred of at least 90% and in particular
preferred of at least 95% identity to SEQ ID NO:5.
The term "identity" means that the sequence of the original murine
sequence and the sequence of the humanized construct are compared
to each other. An identity of for example 90% means that 90% of the
amino acids are at the corresponding amino positions identical in
the original mouse sequence and in the humanized sequence.
The isolated monoclonal antibodies or antigen-binding portions
thereof of the present invention can preferably be used in
pharmaceutical compositions which comprise a pharmaceutically
active amount of the antibody or antigen-binding portion thereof
together with additives suitable for the application to a patient
whereby intraperitoneal application is especially preferred. The
pharmaceutical compositions of the present invention can preferably
be used for the inhibition of inflammation.
It turned out that the monoclonal antibodies or antigen-binding
portions thereof according to the present invention can preferably
be used in the treatment of inflammatory complications following
myocardial infarction. In such complications, which occur
frequently after myocardial infarction inflammatory leukocytes
attracted to the area which is affected by the myocardial
infarction cause and contribute to an inflammatory response that
aggravates wound healing and may inhibit the recovery after
myocardial infarction. In such embodiments the antibodies and
antigen-binding portions thereof according to the present invention
are preferably used.
The inhibition of inflammation by anti-M7 or the derivatives
derived therefrom provides several advantages over a conventional
anti-Mac-1 therapy. It has been observed that mice treated with
formerly known anti-Mac-1 antibodies showed increased mortality
compared to control mice. These data confirm previous studies in
which Mac-1 deficient mice were not protected from bacterial
sepsis, an effect most likely caused by the inability to bind
complement factors and promote clearance of bacterial particles
e.g. by C3bi-mediated phagocytosis.
Previous epitope mapping studies have revealed and located the
binding of C3bi to the residues P.sup.147-R.sup.152,
R.sup.201-K.sup.217, and K.sup.245-R.sup.261 within the
.alpha..sub.M I-domain, demonstrating a binding epitope that is
distinct from the binding sequence required for CD40L
(E.sup.162-L.sup.170). It has been shown that mice treated with
anti-M7 show increased survival compared to anti-Mac-1 and
IgG-control treated mice, indicating that anti-M7 does not only
lack detrimental properties, but induces protective effects.
It is assumed that suppression of pro-inflammatory leukocyte
adhesion in the peritoneum helps to slow-down the overwhelming
pro-inflammatory response accompanying the initial attempt to
remove and fight the bacterial invasion. It is recognized that the
balance between protective and disease aggravating pathways is
disturbed in many conditions and might potentially been shifted to
the protective side by limiting leukocyte recruitment. This
hypothesis is further supported by the fact that anti-M7 protected
from pro-inflammatory cytokine levels in plasma compared with
control animals, while anti-Mac-1 raised cytokine levels. Thus, the
reduction in cytokine levels may be secondary to diminished
leukocyte activation and activation in target tissues. Indeed,
intimal mononuclear cells produce pro-inflammatory cytokines, such
as TNF.alpha., IL-1, IFN.gamma. as well as anti-inflammatory
mediators IL-10. In plasma, mice challenged with TNF.alpha. and
treated with anti-M7 antibody showed a reduction of the
pro-inflammatory cytokines IL-6, TNF.alpha. and MCP-1, while
anti-Mac-1 induced enhanced cytokine expression.
However, treatment with other anti-Mac-1 antibodies, such as the
clone M1/70 (which is used as control), might not entirely reflect
the genetic knock-out. It is noteworthy to mention that M1/70
induces a strong pro-inflammatory response in Mac-1 expressing
cells, in particular in macrophages, and elevates cytokine
expression. The latter is also confirmed by our results,
demonstrating that a single injection of anti-Mac-1 results in
strongly up-regulated cytokine plasma levels, likely affecting
wound healing. It has been suggested that over-stimulation as
provided by M1/70 could represent a feasible strategy to resolve
inflammation by activation of apoptotic pathways. Indeed, it has
previously been shown that apoptosis of cells resident in the
peritoneal cavity was enhanced after a single injection of
anti-Mac-1 clone M1/70. This could potently support anti-Mac-1's
effect in decreasing peritoneal cell accumulation. However, an
apoptosis inducing therapy, accompanied by a cytokine-storm is
likely unfavorable in the clinical practice.
Mac-1 supports interaction to multiple other molecules and more are
likely of not been discovered so far. More than 40 different
protein interactions have been described, but molecular binding
properties of only some of these is known. Therefore it is not to
exclude that the binding site of CD40L is shared by other ligands
as wells. However, the data presented herein unveil and confirm
previous suggestions that CD40L binding to Mac-1 does not share
many features with binding properties to other conventional
ligands: (1) While binding epitopes identified for fibrinogen and
other ligands show overlapping regions, the EQLKKSKTL (SEQ ID NO:9)
motif within Mac-1's I-domain is not involved in binding of
alternative ligands, (2) neither CD40L itself, nor anti-M7 did
induce integrin outside-in signaling, while this feature of
integrin physiology has been considered as paradigm in integrin
ligand binding so far, (3) CD40L's interaction with Mac-1 does not
expand on immune or haemostatic function, while most of Mac-1
ligands, such as Fibrinogen, are involved in multiple of those
pathologies.
The data presented herein propose that the interaction of CD40L
with Mac-1 is primarily required for firm adhesion of inflammatory
leukocytes, presumably of granulocytes in a variety of inflammatory
pathologies. The results do not rule out, but emphasize that immune
function, haemostatic parameters and regenerative response do not
involve binding of CD40L to Mac-1.
It has been shown previously that treatment with the specific
inhibitor of the CD40L/Mac-1 interaction, cM7, attenuates
inflammatory leukocyte recruitment in a model of intravital
microscopy in inflamed cremaster venules, and in a model of sterile
peritonitis. It is demonstrated that treatment with either the full
IgG antibody anti-M7 or F.sub.ab fragments thereof, directed
against the CD40L binding site on Mac-1, significantly reduces
leukocyte adhesion. Interestingly, the inhibitory efficiency of
anti-M7 is comparable to that of anti-Mac-1 treatment, suggesting
the CD40L/Mac-1 interaction as instrumental for leukocyte
recruitment. This does not falsify previous reports, but does
extend the repertoire of Mac-1's ligands expressed on the
endothelium, ICAM-1 and RAGE, by CD40L. In this regard, it is
plausible that patterns of counter-receptor binding depend on
pathologies and the inflammatory burden. Thereby, it is either
possible that the interaction of CD40L and Mac-1 is disease
specific and regulated by either expression of endothelial CD40L,
by conformational change of Mac-1 or that some pathologies are more
dependent on leukocyte invasion than others. For example,
atherosclerosis--a disease in which myeloid cell recruitment is
needed at least in early stages of disease--was strongly
susceptible to blockade of the CD40L/Mac-1 interaction, while
neo-intima formation after a wire injury was not inhibited by
blocking CD40L/Mac-1, but by anti-Mac-1 or in Mac-1 knock-out
mice.
The data obtained in the course of the present invention show that
anti-M7 was most effective in blocking the interaction to activated
Mac-1, but not to non-activated Mac-1. This proposes that the
interaction may play a more important role in pathologies
associated with a higher inflammatory burden, rather than under
baseline conditions.
Also, it remains to be answered whether an antibody such as anti-M7
can actively modulate or conserve different conformations of the
integrin as previously proposed. This could explain that only the
permanently-activated integrin, but not the integrin in native
condition was targeted as the data show. However, for the
determination of the exact binding properties a more detailed
structural analyses may be helpful.
Finally, it cannot be excluded that CD40L/Mac-1 interaction may be
responsible for the egress and mobilization of monocyte from the
bone marrow or the spleen as previously suggested. As observed
herein, inflammatory monocytosis during sepsis could be completely
reversed by anti-M7 treatment. Whether this is caused by impaired
monocyte reservoirs, e.g. by impaired migration to the spleen,
shall be determined in further experiments.
The antibodies of the present invention follow a strategy to
selectively target the EQLKKSKTL (SEQ ID NO:9) binding motif,
representing CD40L's binding site within the Mac-1 I-domain, by a
monoclonal antibody anti-M7. This antibody is highly selective for
the targeted binding site, does not interfere with alternative
binding partners, and--in contrast to conventional anti-Mac-1
antibodies--does not affect haemostasis, host defense and wound
healing. In preferred embodiments the antibodies of the present
invention do not interfere with alternate binding partners and are
therefore highly selective for the targeted binding site. The
proposed ligand-targeted anti-integrin therapy is superior to an
unselective approach and represents an advantage to refine and
adjust anti-integrin therapy against inflammatory disease.
The results, experiments and advantages obtainable by the present
invention are summarized in the Figures and the Examples. Figures
and Examples show preferred embodiments of the present invention,
in particular the most preferred antibody anti-M7, but it should be
understood that Figures and Examples should not be considered as
limiting the present invention.
The preferred embodiments of the invention are shown in the Figures
and in the Examples:
BRIEF DESCRIPTION TO DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIG. 1 shows that A mouse monoclonal antibody raised against the
CD40L binding site within human Mac-1, anti-M7, is effective in
targeting the human integrin. The peptide sequence M7 within the
Mac-1, required for binding of CD40L, is a highly conserved binding
motif between the human (SEQ ID NO:9) and murine (SEQ ID NO:10)
integrin (FIG. 1A).
Furthermore, FIG. 1A shows the peptide M1 of human origin (SEQ ID
NO:14) and the corresponding peptide M1 derived from Mus musculus
having SEQ ID NO:15. The human peptide having the designation M8
corresponds to SEQ ID NO:13 and the peptide M8 derived from Mus
musculus has SEQ ID NO:16.
Antibody anti-M7 generated by immunization of mice with the binding
peptide VMEQLKKAKTLMQ (SEQ ID NO:11) coupled to diphtheria toxoid
bound to a CHO cell line over-expressing native (WT) and
permanently activated Mac-1 (del), but not to control CHO cells in
western blot (FIG. 1B).
Specific binding of the antibody anti-M7 to the immobilized
peptides M7 (EQLKKSKTL) (SEQ ID NO:9), sM7 (KLSLEKQTK) (SEQ ID
NO:12), and M8 (EEFRIHFT) (SEQ ID NO:13) was tested in a solid
phase binding with immobilized peptides (FIG. 1C).
Binding was quantified by binding of biotinylated anti-mouse IgG
and color reaction after incubation with HRP-coupled streptavidin.
Specific binding was calculated by subtraction of binding of mouse
IgG to the peptides. Anti-M7 was coupled with the fluorochrome
Alexa647 and binding to human leukocyte subsets was quantified in
FACS. Alexa647 Isotype antibody served as control (FIG. 1D).
FIG. 2 shows that Anti-M7 selectively blocks the interaction of
permanently activated Mac-1 with CD40L, but not of the native
integrin or to alternative Mac-1 ligands. CHO-cells over-expressing
the permanently activated Mac-1 mutant (Mac-1-del) adhered to
immobilized CD40L in a static adhesion assay (FIG. 2A, 2B).
Cells were incubated with anti-M7 or the human pan-I-Domain
blocking reference clone 2LPM19c 15 min prior to adhesion.
Alternatively, adhesion of the native, non-activated Mac-1 integrin
was tested (FIG. 2C). To exclude unspecific Fc-mediated
interaction, F.sub.ab-fragment preparation of anti-M7 or anti-Mac-1
were used as inhibitor (FIG. 2D).
To test whether anti-M7 is specific for CD40L, a panel of classical
Mac-1 ligands were separately immobilized and adhesion of
permanently activated Mac-1 CHO cells was quantified in the
presence of anti-M7 or pan I-Domain blocking anti-Mac-1 (FIG.
2E).
FIG. 3 shows that Anti-M7 does not induce integrin outside-in
signaling, while conventional anti-Mac-1 antibodies induce
activation of MAP-kinases and inflammatory cytokine expression in
vitro and in vivo.
Murine macrophages were isolated by injection of 4% thioglycollate
in the peritoneum of C57Bl/6 mice and incubation for 72 hours.
Peritoneal cells were collected by peritoneal lavage, FACS analysis
confirmed purity of >90 percent F4/80.sup.+ macrophages.
Macrophages were cultured in 5% FCS RPMI overnight and stimulated
with 10 .mu.g/ml of mouse IgG, anti-human Mac-1 (clone 2LPM19c),
anti-mouse Mac-1 (clone M1/70) or anti-M7 for 30 min. Cells were
lysed and phosphorylated ERK1/2, Nf.kappa.B and p38 were visualized
by western blot (FIG. 3A), and the ratio of phosphorylated
fractions was calculated (FIG. 3B). Values were calculated as
relative arbitrary units (AU) normalized to signal of cells
stimulated with saline alone. Mac-1 antibody clones were injected
i.p. in mice and serum concentration of IL-6, TNF.alpha., and MCP-1
was measured by cytometric bead array 4 hours after injection (FIG.
3C). Anti-Mac-1 clone 1/70 was used as control.
FIG. 4 shows that treatment with anti-M7 prevents inflammatory
leukocyte recruitment in vitro and in vivo and decreases
inflammatory cytokine expression. Murine RAW-cells were allowed to
adhere on isolated and TNF.alpha.-primed murine endothelial cells
in vitro in a flow chamber assay. Number of adhering cells was
quantified in the presence of an anti-mouse IgG or anti-M7 antibody
(FIG. 4A). C57Bl/6 mice were injected with 200 ng TNF.alpha. i.p.
to induce peritoneal and mesenteric inflammation. Simultaneously,
either IgG isotype control or anti-mouse anti-Mac-1 (clone M1/70)
F.sub.ab-fragment preparations were injected. Leukocyte recruitment
to inflamed mesenteric venules was monitored by intravital
microscopy 4 hours after injection (FIG. 4B). Number of adhering
and rolling leukocytes were quantified, as well as leukocyte
rolling velocity, displayed as cumulative frequency (FIG. 4C-E).
Mice expressing GFP in monocytes (CX3CR1-GFP) were subjected to
intravital microscopy in the presence of IgG or anti-M7 F.sub.ab
preparations (FIG. 4F). Migrated monocytes (white arrows) were
quantified in the para-vascular space in the viewing field (FIG.
4G). Plasma cytokine levels in mice subjected to intravital
microscopy after IgG or anti-M7 F.sub.ab treatment were assessed by
CBA bead array (FIG. 4H).
FIG. 5 shows that Anti-M7 does not affect venous thrombosis and
platelet effector function in vivo. Venous thrombosis was induced
in mesenteric venules of C57Bl/6 mice by ferric chloride. Thrombus
formation was visualized by in vivo rhodamine staining in
intravital microscopy (FIG. 5A). Time to thrombus-occlusion of the
vessel and rate of emboli (/min) was monitored and quantified (FIG.
5B, 5C). Mice were treated with either F.sub.ab-preparation of
mouse IgG, anti-M7 or anti-Mac-1 (50 .mu.g) by intraperitoneal
injection 15 min prior to thrombus induction. Formation of
platelet-monocyte aggregates was quantified by detection of
CD41.sup.+ monocytes in flow cytometry after treatment with
anti-Mac-1 antibody clones (FIG. 5D).
FIG. 6 shows that specific inhibition of Mac-1's interaction to
CD40L, but not to other ligands, improves skin wound healing.
Aseptic skin wounds were induced by a 4-mm biopsy punch after
injection of anti-Mac-1 or anti-M7 F.sub.ab preparations. After 6
days skin wounds were photographed (FIG. 6A) and wound area was
calculated (FIG. 6B).
FIG. 7 shows that Anti-M7 improves host defense, bacterial
clearance, and inflammation during bacterial sepsis, while
unspecific blockade of Mac-1 potentiates bacteremia in mice. To
test whether blockade of Mac-1 or specifically of the CD40L binding
site affects host defense and inflammation during bacterial sepsis,
coecal-ligation and puncture sepsis (CLP) was induced. 20 hours
after CLP procedure inflammatory and patrolling monocytes
circulating in blood were quantified by flow cytometry (FIG. 7A).
Granulocytes (F4/80.sup.-Gr-1.sup.+) invading into the peritoneal
cavity were identified by flow cytometry (FIG. 7B) and total
numbers were calculated (FIG. 7C). Levels of the acute phase
protein SAA (FIG. 7D) and of bacterial LPS titers (FIG. 7E) were
quantified in plasma. Accumulation of granulocytes in kidney
parenchyma was determined by staining against DAP and Ly6G (FIG.
7F) and quantified as ratio of granulocytes/total cell nuclei (FIG.
7G).
FIG. 8 shows that Anti-M7 improves, while anti-Mac-1 decreases,
survival during CLP-sepsis. Coecal-ligation and puncture sepsis
(CLP) was induced. To assess if treatment with Mac-1 antibody
clones affects survival, mice were treated by intraperitoneal
injection with either anti-Mac-1 or anti-M7 F.sub.ab preparations
at 0, 48, and 96 hours after induction of CLP sepsis. Relative
survival was calculated and displayed as Kaplan-Maier survival
cure.
FIG. 9 shows that treatment with Anti-M7 blocks inflammatory
leukocyte infiltration in the injured myocardium following
myocardial infarction. Myocardial infarction was induced by a
surgical ligation of the left anterior descending coronary artery
(LAD). Leukocytes infiltrating the infarcted myocardium were
quantified by flow cytometry in digested hearts after myocardial
infarction. Anti-M7 decreased the infiltration with monocytes and
neutrophils and attenuated heart failure as assessed by
echocardiography.
The results summarized in the Figure were obtained in the following
examples:
EXAMPLE 1
Male mice on a C57BL/6N background received a standard chow diet.
All mice were maintained under standardized conditions (12-hour
light, 12-hour dark cycle) and had access to food and water ad
libidum. At the age of 8 weeks, mice were subjected to intravital
microscopy, wound healing or CLP sepsis as indicated. Treatment
with antibodies was performed by intraperitoneal injection in the
indicated concentration at a volume of 100 uL per injection. In
some intravital experiments, GFP-transgene animals under the
control of CXCR3-promoter (CXCR3-GFP) were used to track
leukocytes. All experimental protocols were approved by the animal
ethics committee of the Alfred Medical Research and Education
Precinct (AMREP), Melbourne, Australia and the local animal ethics
committee at the University of Freiburg. All procedures were
carried out in accordance with institutional guidelines.
An antibody specific for a peptide corresponding to Mac-1 I-domain
sequence V160-S172 was obtained by immunizing mice with the peptide
C-VMEQLKKSKTLFS-NH2 (SEQ ID NO:17) coupled to diphtheria toxoid
(Monash Antibody Technologies Facility, Monash University,
Melbourne, Australia). Solid phase binding assays was employed to
screen binding of sera to the immobilized peptide M7. Among
different clones binding with high affinity to M7, the preferred
clone RC3 (termed anti-M7) was further characterized.
EXAMPLE 2
A mouse monoclonal antibody raised against the CD40L binding site
within human Mac-1, anti-M7, is effective in targeting the human
integrin.
It has previously been shown that CD40L selectively binds to the
EQLKKSKTL (SEQ ID NO:9) motif within the major Mac-1 ligand-binding
domain. To obtain a specific inhibitor of the human binding site,
mice were immunized with the human peptide V160-S172 containing the
binding peptide M7. Interestingly, the M7 sequence was highly
conserved between the human and murine protein sequence (FIG. 1A).
Among several hybridoma clones with high-affinity binding of the
according supernatant to the immobilized peptide M7 in a
solid-phase binding assay, clone RC3 (mouse IgG2b.kappa.) showed
specific inhibition of Mac-1-CD40L binding, but not of the
interaction to other ligands. This antibody clone, subsequently
termed anti-M7, bound to a CHO cell line over-expressing native
(WT) and permanently activated Mac-1 (del), but not to control CHO
cells in western blot (FIG. 1B), confirming successful binding to
the target protein.
Moreover, anti-M7 bound to the immobilized peptides M7 (EQLKKSKTL)
(SEQ ID NO:9), but not to the control peptides scrambled sM7
(KLSLEKQTK) (SEQ ID NO:12) or the peptide M8 (EEFRIHFT) (SEQ ID
NO:13) in a solid phase binding (FIG. 1C), indicating that anti-M7
specifically binds to the immunized peptide. To test binding of
anti-M7 to Mac-1 expressing human cells, we coupled the antibody
with the fluorochrome Alexa647 and quantified binding to human
leukocyte subsets in flow cytometry. Interestingly anti-M7 showed
concentration-dependent binding to human leukocytes expressing
Mac-1, such as monocytes and neutrophils, but not to lymphocytes as
expected (FIG. 1D). Binding of anti-Mac-1 clone M1/70 served as
control and showed the same binding properties with highest binding
to myeloid cells. These findings demonstrate that the binding
sequence M7 within the human Mac-1 I-domain is accessible to
binding with the monoclonal antibody anti-M7. Further DNA
sequencing revealed CDRs and exact protein sequence of anti-M7
variable regions of heavy and light chain. This is shown in Table
1:
TABLE-US-00001 TABLE 1 Protein sequence of anti-M7 variable regions
Light chain DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWHQQKPDGTIKRLLYS
TSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYAISPPTFGG GTKLEIK (SEQ ID
NO: 1) Heavy chain
QVTLKESGPGILQTSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTTDTATYYCALN
YYNSTYNFDFWGQGTTLTVSS (SEQ ID NO: 5) Position of CDR 1,2,3 is
underlined
EXAMPLE 3
Specific binding of the antibody anti-M7 to the immobilized
peptides M7 (EQLKKSKTL) (SEQ ID NO:9), sM7 (KLSLEKQTK) (SEQ ID
NO:12), and M8 (EEFRIHFT) (SEQ ID NO:13) was tested in a solid
phase binding with immobilized peptides in 96-well ELISA plates
(Nunc). Binding of anti-M7 was detected by addition of biotinylated
anti-mouse IgG and subsequent color reaction after incubation with
HRP-coupled streptavidin and TMB-substrate. Specific binding was
calculated by subtraction of binding of mouse IgG to the peptides.
To test binding of the antibody anti-M7 to human leukocytes,
anti-M7 was labeled with Alexa Fluor 647 according to the
manufacturers protocols (Monoclonal Antibody Labeling Kit, Life
Technologies). Human leukocytes were isolated from healthy donors
by centrifugation and Red Blood Cell lysis-Leukocytes were
stimulated with PMA (200 ng/ml), incubated with anti-M7-Alexa 647
(1 .mu.g and 5 .mu.g) and antibody binding was quantified by flow
cytometry.
It was found that anti-M7 is a ligand- and activation specific
inhibitor of Mac-1's interaction with CD40L.
To test whether anti-M7 is able to functionally block the
interaction of Mac-1 and CD40L, the adhesion of CHO-cells
over-expressing a permanently activated Mac-1 mutant (Mac-1-del) to
immobilized CD40L was tested in a static adhesion assay.
Interestingly, anti-M7 blocked the cell adhesion by 65.6.+-.7.2%,
an effect nearly as strong as the anti-human pan-I-Domain blocking
reference clone 2LPM19c (inhibition by 92.7.+-.2.0%, FIG. 2A, B).
In the experiment a concentration of 10 .mu.g/ml was used. It can
be concluded therefrom that in general concentrations of the
antibody ranging from 1 to 50 .mu.g/ml and preferably from 5 to 20
.mu.g/ml are used. Most interestingly, in contrast to the reference
anti-Mac-1 antibody, anti-M7 did not block adhesion of CHO cells
expressing the native, non-activated Mac-1 integrin (FIG. 2C),
indicating that blockade by anti-M7 was specific to high-affinity
conformation of the integrin. Moreover, inhibition by anti-M7 was
not restricted to human proteins, since interaction of murine
macrophages and murine CD40L was significantly blocked by anti-M7.
Furthermore, blocking by anti-M7 was not unspecifically caused by
the F.sub.c-fragments of the antibody, since F.sub.ab-fragment
preparations of anti-M7 or anti-Mac-1 were as effective as the
whole antibody preparation (FIG. 2D). Different ligands can bind to
separate or overlapping binding regions within the Mac-1 I-domain.
To test whether anti-M7 is specific for the CD40L binding epitope,
a panel of classical Mac-1 ligands, such as Fibrinogen, ICAM-1,
NIF, heparin, and RAGE was separately immobilized and binding of
Mac-1-del cells was tested in the presence of anti-M7 and
anti-Mac-1 (FIG. 2E). Notably, anti-Mac-1 blocked each of the
interactions, while blocking capacity of anti-M7 was restricted to
CD40L. These data unveil that anti-M7 is an effective and specific
inhibitor of the CD40L/Mac-1 interaction.
EXAMPLE 4
Murine peritoneal macrophages were obtained as described above.
Flow cytometry revealed that the majority (>90%) of PECs were
positive for the macrophage marker F4/80. After overnight
starvation macrophages were stimulated with the indicated
antibodies against Mac-1 in a concentration of 10 .mu.g/ml for 30
minutes. After the indicated time points, cells were lysed,
proteins were separated by SDS-PAGE and blotted to polyvinylidene
difluoride membranes. Total protein and the phosphorylated fraction
of NF.kappa.B, ERK1/2 and p38 were detected by specific antibody
binding in western blot (Cell Signaling). The ratio of
phosphorylated fractions was calculated and expressed as relative
arbitrary unit (AU) normalized to signal of cells stimulated with
saline alone.
The test results show that anti-M7 does not induce integrin
outside-in signaling, while conventional anti-Mac-1 antibodies
induce activation of MAP-kinases and inflammatory cytokine
expression in vitro and in vivo.
Conventional anti-Mac-1 antibodies induce activation of the
integrin, termed outside-in-signaling mediated by downstream
activation of MAP-kinases, such as ERK and p38 upon ligand and
antibody binding. It has previously been shown that CD40L is a
biased agonist not inducing outside-in signaling events upon
binding. To test whether anti-M7 would induce cell activation,
thioglycollate-elicited peritoneal macrophages from male, 8 week
old C57Bl/6 mice were collected. After overnight starvation in 5%
FCS containing RPMI, the macrophages were stimulated with 10
.mu.g/ml of either mouse IgG, anti-human Mac-1 (clone 2LPM19c),
anti-mouse Mac-1 (clone M1/70) or anti-M7 for 30 min. Anti-Mac-1
treatment induced phosphorylation of ERK and p38 as quantified by
an elevated ratio of the phosphorylated epitopes in western blot
(FIG. 3A), while anti-M7 had no effects, indicating that the
binding epitope targeted by anti-M7 is not involved in outside-in
signaling (FIG. 3B). To assess whether this effect is relevant for
an in vivo treatment, Mac-1 antibody clones were injected i.p. in
mice and serum concentration of IL-6, TNF.alpha., and MCP-1 were
quantified 4 hours after injection. Surprisingly, the Mac-1
reference clone M1/70 (control) strongly elevated cytokine levels,
while anti-M7 did not (FIG. 3C). In accordance, levels of
pro-inflammatory cytokines increased in in vitro culture of
macrophages after antibody stimulation. These findings indicate
that anti-M7 is targeting an epitope not causing unwanted
outside-in signaling during integrin blockade.
EXAMPLE 5
Before enzymatic digestion, the antibody was dialyzed in a
SnakeSkin Dialysis Tubing 10k MWCO against PBS overnight at
4.degree. C. Immobilized papain was used to prepare F.sub.ab
fragments from anti-M7, anti-Mac-1 (clone M1/70) and an IgG isotype
control as indicated according to the manufacturer's instructions
(Pierce F.sub.ab Preparation Kit, Thermo Scientific). Briefly,
F.sub.ab-fragments were generated in the presence of 25 mM cysteine
for 3 h at 37.degree. C., followed by purification on NAb Protein A
Spin Columns. Purity of F.sub.ab-fragments was evaluated on
SDS-PAGE.
96-well plates (Nunc) were coated with sCD40L (10 .mu.g/ml) and
incubated with CHO-cells expressing constitutively activated Mac-1.
Cells were pre-incubated with blocking antibodies (10 .mu.g/mL) as
indicated and allowed to adhere for 50 minutes. Adhering cells were
counted after repeated washing with PBS. For dynamic adhesion
assays, human umbilical endothelial cells (HUVECs) were grown to
confluency in 35 mm cell culture dishes, stimulated with TNF.alpha.
overnight and placed in a parallel flow chamber system (Glycotech).
Number of adhering cells was quantified at the indicated shear rate
in the presence of the indicated antibodies (10 .mu.g/mL).
For intravital microscopy mice received an intraperitoneal
injection of 100 .mu.g of antibodies or 50 .mu.g of
F.sub.ab-fragments i.p. After 15 minutes mice were injected i.p.
with 200 ng murine TNF.alpha. (R&D Systems). Surgery started 4
hours after TNF.alpha. administration. Briefly, mice were
anesthetized by intraperitoneal injection of ketamine hydrochloride
(Essex) and xylazin (Bayer, Leverkusen, Germany). The mesentery was
exteriorized and placed under an upright intravital microscope
(AxioVision, Carl Zeiss). Videos of rolling and adhering in
mesenteric venules were taken after retro-orbital injection of
rhodamine. Rolling leukocyte flux was defined as the number of
leukocytes moving at a velocity less than erythrocytes. Adherent
leukocytes were defined as cells that remained stationary for at
least 30 seconds.
Flow cytometry: Peritoneal exudate cells (PECs) and blood
leukocytes were obtained as described below. Remaining red blood
cells were removed by incubation with a red blood cell lysing
buffer (155 mM NH4Cl, 5.7 mM K2HPO4, 0.1 mM EDTA, pH7.3). Cells
were washed in PBS, and Fc-Receptors were blocked by anti-CD16/CD32
(eBioscience) for 10 minutes on ice. Cells were then labeled with
the indicated antibodies before quantification with a flow
cytometer (FACS Calibur, BD Biosciences). All antibodies were
obtained from eBioscience. Distinct leukocyte populations were
identified upon cell surface expression of the indicated antigens:
granulocytes (Gr-1.sup.+F4/80.sup.-CD11b.sup.+CD115), macrophages
(F4/80.sup.+CD11b.sup.+CD115.sup.-), inflammatory monocytes
(CD11b.sup.+CD115.sup.+Gr-1.sup.+4/80.sup.-), non-inflammatory
monocytes (CD11b.sup.+CD115.sup.+Gr-1.sup.-F4/80.sup.-).
Isolation and cultivation of murine peritoneal macrophages:
Antibodies were injected i.p. 30 min before WT mice received an
injection of 2 mL of 4% thioglycollate broth (Sigma). A peritoneal
lavage was performed after 72 hours. Peritoneal exudate cells
(PECs) were quantified and characterized by FACS as described
above. In CLP experiments a peritoneal lavage was performed 20
hours after surgery.
It could be shown that treatment with anti-M7 prevents inflammatory
leukocyte recruitment in vitro and in vivo and decreases
inflammatory cytokine expression.
Mac-1 is a powerful adhesion factor, likely mediating its adhesive
function through interaction with different ligands expressed at
the endothelium, including ICAM-1, RAGE, and CD40L. To test if
anti-M7 blocks cellular adhesion, murine monocyte-like RAW-cells
were allowed to adhere on isolated and TNF.alpha.-primed murine
endothelial cells in vitro in a flow chamber assay. Number of
adhering cells decreased after incubation with anti-M7, indicating
that CD40L/Mac-1 interaction is required for leukocyte arrest (FIG.
3A). To test for relevance of these findings in vivo,
F.sub.ab-fragment preparation of anti-M7 and an according isotype
were injected i.p. prior to intravital microscopy (FIG. 4B).
Leukocyte recruitment to inflamed mesenteric venules was monitored
after simultaneous stimulation with TNF.alpha. for 4 hours to
induce inflammatory leukocyte recruitment. Consistently with our in
vitro results, we observed that the number of adhering (FIG. 4C),
but not of rolling leukocytes (FIG. 4D) was reduced after anti-M7
injection. In accord, leukocyte rolling velocity, displayed as
cumulative frequency, was not changed (FIG. 4E), indicating that
firm adhesion, but not rolling properties of leukocyte is blocked
by anti-M7. To exclude that anti-M7 induces leukocyte depletion we
injected anti-M7 or an according isotype control i.p., and
quantified leukocyte populations. Of note, no changes were observed
in both groups. To test if impaired monocyte arrest would affect
down-stream effects, such as transmigration, mice expressing GFP in
monocytes (CX3CR1-GFP) were subjected to intravital microscopy in
the presence of IgG or anti-M7 F.sub.ab preparations after a
TNF.alpha. challenge for 4 hours (FIG. 4F). In accordance, we
observed that anti-M7 treated animals showed lower numbers of
monocytes migrated to the perivascular space (FIG. 4G). Finally, we
observed that plasma levels of the pro-inflammatory cytokines
TNF.alpha., IL-6, and MCP-1 were significantly reduced in mice
subjected to intravital microscopy after anti-M7 F.sub.ab treatment
compared with IgG F.sub.ab treated control animals (FIG. 4H). These
results clearly indicate that leukocyte adhesion proceeds by the
interaction of CD40L and Mac-1 and that this interaction can be
functionally blocked by anti-M7 antibody.
EXAMPLE 6
It has also been shown that anti-M7 does not affect venous
thrombosis and platelet effector function in vivo.
Mac-1 participates in haemostasis and thrombus formation,
presumably by its interaction to the platelet glycoprotein
GP1b.alpha.. Also, CD40L stabilizes thrombi and its therapeutic
inhibition raises thromboembolic complications. To exclude that an
antibody according to the invention would induce unwanted thrombus
destabilization, venous thrombosis was induced in mesenteric
venules of C57Bl/6 mice by ferric chloride. Thrombus formation was
visualized by in vivo rhodamine staining in intravital microscopy
(FIG. 5A). As described previously, inhibition of Mac-1 by an i.p.
injected F.sub.ab-fragment prolonged vessel occlusion time and
increased the release of thrombotic emboli (FIG. 5B, C), confirming
that Mac-1 is needed to stabilize thrombi. However, inhibition by
anti-M7 did not cause significant changes in vessel occlusion time
or release of thrombotic emboli, proposing that participating
pathways were not affected. Accordingly, formation of
leukocyte-platelet aggregates was diminished by unspecific blockade
of Mac-1, but not by specific inhibition of the CD40L/Mac-1
interaction (FIG. 5D). These data propose that anti-M7 is likely
not inducing unwanted effects on the haemostatic system.
EXAMPLE 7
Interaction of Mac-1 to CD40L, but not to other ligands, improves
skin wound healing. Leukocyte engagement is a critically step in
wound healing and delayed wound healing has been reported in Mac-1
null mice. To test whether these effects are mediated by Mac-1's
interaction to CD40L, we treated C57Bl/6 mice with i.p.-injections
of F.sub.ab-fragments of either anti-M7, anti-Mac-1 or an according
isotype control directly after induction of 4 mm dorsal skin
wounds. Interestingly, during the time course of the experiment
delayed wound healing in anti-Mac-1 treated mice was not detected.
However, skin wounds tent to close faster in Kaplan-Maier wound
closure analysis in anti-M7 treated mice and demonstrated a smaller
wound surface 6 days after wound induction (FIG. 6A,B). This
indicates that specific inhibition of the CD40L/Mac-1 interaction
does not affect, but instead seems to exhibit protective effects on
skin wound healing.
EXAMPLE 8
Unselective inhibition of Mac-1 aggravates, while specific blockade
of its interaction to CD40L improves bacterial clearance,
inflammation, and survival during bacterial sepsis.
It has recently been shown that mice with a genetic deficiency of
Mac-1 demonstrated decreased survival during bacterial sepsis,
highlighting the potential role of the leukocyte integrin in host
defense and clearance of bacteria. To elucidate whether
ligand-specific blockade of Mac-1 and CD40L is rather beneficial
during bacterial sepsis, a model of coecal-ligation and puncture
sepsis (CLP) was performed. 20 hours after CLP procedure
inflammatory and patrolling monocytes circulating in blood and
basic inflammatory parameters were quantified. Interestingly, CLP
induced a strong mobilization of inflammatory Gr-1.sup.+ monocytes
to the circulation, reaching a percentage of the inflammatory
subset of about 82.4.+-.4.6% of all monocytes in IgG
F.sub.ab-fragment treated mice. This response was not affected by
F.sub.ab anti-Mac-1 treatment (77.4.+-.6.0%), but nearly reversed
by F.sub.ab anti-M7 treatment (56.8.+-.3.7%, FIG. 7A). During CLP,
myeloid cells populate the peritoneal cavity. Granulocytes
(F4/80.sup.-Gr-1.sup.+) invading the peritoneal cavity were
identified by flow cytometry (FIG. 7B). Both, anti-Mac-1 and
anti-M7, strongly reduced granulocyte accumulation by 59.9.+-.12.2%
and 73.8.+-.7.1% for anti-Mac-1 and anti-M7, respectively (FIG.
7C). The anti-inflammatory effect of anti-M7 treatment was further
reflected by a strong decrease of the acute-phase protein SAA by
63.4.+-.19.7% (FIG. 7D). Notably, anti-M7 improved bacterial
clearance in the plasma, while anti-Mac-1 worsened bacterial load
in both, plasma and the peritoneal cavity (FIG. 7E). During CLP,
accumulation of neutrophils is observed in the periphery, such as
the kidney and lung. To quantify granulocyte trafficking to the
spleen, ICH was performed against the granulocyte marker Ly6G in
kidney sections (FIG. 7F). Notably, both anti-integrin therapies
prevented neutrophil accumulation with a stronger effect in
anti-Mac-1 treated animals (FIG. 7F). Finally, it was assessed if
the new ligand-specific approach according to the invention is
beneficial in surviving sepsis. Therefore, CLP was induced and
animals were subsequently treated with F.sub.ab-preparations of
IgG, anti-Mac-1 and anti-M7 at 0, 48, and 96 hours after induction
of CLP operation.
Survival rate was calculated employing Kaplan-Maier analysis and
log-rank testing. Animals treated with anti-Mac-1 showed
significantly decreased mean survival compared to IgG-control
treated animals (0% vs. 6.7% after 169 hours after CLP-induction
for anti-Mac-1 and IgG, respectively). Notably, anti-M7 treated
showed a survival rate of 40.0% at the end of the study (FIG. 8),
demonstrating that ligand-directed therapy is superior to
unspecific inhibition.
EXAMPLE 9
Treatment with anti-M7 improves the infiltration with inflammatory
leukocytes in the injured myocardium following myocardial
infarction. Accumulation of inflammatory leukocyte occurs after
myocardial infarction within days. Inflammatory leukocyte recruited
to the infarcted heart cause an inflammatory response that
aggravates wound healing and drives heart failure after myocardial
infarction. Inhibition of leukocyte infiltration has been proposed
to represent a therapeutic strategy, but not such strategy is
available. After induction of myocardial infarction in mice by a
surgical ligation of the left anterior descending coronary artery
(LAD) and treatment with anti-M7 less infiltrating monocytes and
neutrophils, a subclass of inflammatory leukocytes that express
Mac-1, were found in the injured myocardium. As a result, anti-M7
attenuated heart failure.
SEQUENCE LISTINGS
1
171107PRTartificial sequencelight chain 1Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Glu Arg Val Ser Leu
Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30Leu Ser Trp His
Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Leu 35 40 45Tyr Ser Thr
Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60Ser Arg
Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75
80Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ile Ser Pro Pro
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10526PRTartificial sequenceCDR 1 2Gln Glu Ile Ser Gly Tyr1
533PRTartificial sequenceCDR 2 3Ser Thr Ser149PRTartificial
sequenceCDR 3 4Leu Gln Tyr Ala Ile Ser Pro Pro Thr1
55121PRTartificial sequenceheavy chain 5Gln Val Thr Leu Lys Glu Ser
Gly Pro Gly Ile Leu Gln Thr Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys
Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Val Ser
Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His
Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser
Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val65 70 75 80Phe
Leu Lys Ile Thr Ser Val Asp Thr Thr Asp Thr Ala Thr Tyr Tyr 85 90
95Cys Ala Leu Asn Tyr Tyr Asn Ser Thr Tyr Asn Phe Asp Phe Trp Gly
100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser 115
120610PRTartificial sequenceCDR1 6Gly Phe Ser Leu Ser Thr Ser Gly
Met Gly1 5 1077PRTartificial sequenceCDR 2 7Ile Tyr Trp Asp Asp Asp
Lys1 5813PRTartificial sequenceCDR 3 8Ala Leu Asn Tyr Tyr Asn Ser
Thr Tyr Asn Phe Asp Phe1 5 1099PRTartificial sequencepeptide 9Glu
Gln Leu Lys Lys Ser Lys Thr Leu1 5109PRTartificial sequencepeptide
M7 10Glu Gln Phe Lys Lys Ser Lys Thr Leu1 51113PRTartificial
sequencebinding peptide 11Val Met Glu Gln Leu Lys Lys Ala Lys Thr
Leu Met Gln1 5 10129PRTartificial sequencepeptide 12Lys Leu Ser Leu
Glu Lys Gln Thr Lys1 5138PRTartificial sequencepeptide 13Glu Glu
Phe Arg Ile His Phe Thr1 51413PRTartificial sequencepeptide M1
14Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr1 5
101513PRTartificial sequencepeptide M1 15Asn Ile Asp Phe Gln Lys
Met Lys Glu Phe Val Ser Thr1 5 10168PRTartificial sequencepeptide
M8 16Asp Glu Phe Arg Ile His Phe Thr1 51713PRTartificial
sequencepeptide 17Val Met Glu Gln Leu Lys Lys Ser Lys Thr Leu Phe
Ser1 5 10
* * * * *